Functional Characterization Of Heat Tolerant Gene LrgB And Fruit Firmness Related Genes

Tomato(Solanum lycopersicum)is one of the most important vegetables in the world and it plays an important role in vegetable industry of our country.In our study,we researched the stress and softening resistant in tomato respectively,and both made advances.All along,heat resistance research play an important role in tomato scientific research.It has been shown that Heat shock protein 70 KD(Hsp70)plays an important role in heat resistance.In our research,we used Heat shock cognate protein70 KD(Hsc70.1)as bait and acquired the Lrg B protein.Functions of the gene encoding Lrg B in heat resistant,chloroplast development and carbon assimilation partitioning were investigated.The main results were as follows:1.Using yeast two-hybrid and Bi FC assays,we found Sl Lrg B protein interacted with Hsc70.1.There is only one homologus gene exsited in tomato,here designated Sl Lrg B.Lrg B participate in programmed cell death(PCD),in tomato it was significantly down-regulated under heat stress,so we assumed that Sl Lrg B interacts with Hsc70.1 to play roles together in plants.The heat resistance of transgenic lines was also tested and the results showed that the RNAi transgenic lines exhibited much higher heat resistance than WT plants.To our knowledge,this is the first report about Lrg B gene participating in heat stress response in plant.2.The subcellular localization analysis suggested that Sl Lrg B was located in chloroplast.The tissue-specific expression analysis suggested that it only expressed in green tissues.The Sl Lrg B-overexpression seedlings exhibited chlorotic cotyledons in leaves,while Sl Lrg B-interference seedlings led to pale-green leaves phenotype compared with wild type.Both the chlorophyll and carotenoid content were down-regulated in transgenic seedlings,and the maximum photosynthetic efficiency in their leaves were declined.According to this,the transgenic plants showed lower plant height and weight,the pericarp color of MG fruit in OE lines were paler compared with WT and RNAi lines.It declared that Sl Lrg B also regulate the chlorophyll content in fruit.3.Using transmission electron microscope observe the cell and chloroplast,we found that both the chloroplast quantity and the density of thylakoid structure were significantly decreased.Meanwhile,cell quantity was also decreased in OE tissues.We researched the expression levels of genes involved in PCD,and found that these genes had been regulated in both the overexpression and interference plants,the regulatory pattern were opposite.Besides,the starch content was significantly higher and the sucrose content was lower in RNAi lines compared with WT lines,while the OE lines exhibited the contrary behavior.These results illustrated that Sl Lrg B participates in chloroplast development,PCD and carbon assimilation partitioning.4.The expression analysis of Hsc70.1,TPT and 14-3-3 in transgenic lines showed that the abnormity of carbon assimilation partitioning in RNAi lines may due to the change of TPT expression level while heat resistance and PCD in transgenic plants may influenced by the change of Hsc70.1 expression level.Virus induced gene silencing(VIGS)of Hsc70.1 was also performed,the result showed that plants VIGS of Hsc70.1 exhibited leaf chlorosis and growth retardation compared with negative control.It improved that Sl Lrg B should play a role in chloroplast together with Hsc70.1.In tomato fruit softening research,as a kind of climacteric fruit,during the process of fruit maturation,there existed augmented respiration,firmness reduction and increased nutrient composition in tomato fruit.Therefore,excavating the fruit firmness regulation-related genes is significant.In our research,we determined the firmness of red fruit of 269 diverse global collection of tomato accessions at two locations.According to analysed the GWAS result of fruit firmness,we found out an ethylene receptor gene(EIN4-like protein)on chromosome 4.We compared the gene sequence in lower and higher fruit firmness respectively,then we found out there was a single nucleotide polymorphism(A?G)result in an amino acid mutation(Lys?Glu)between them,so we assumed that this gene should has the function of regulating the fruit firmness.From an evolutionary perspective,we considered the genotypic variation should be one of the reason for the change of fruit firmness.